Heitmar et al.1 suggest a new approach called sequential and diameter response analysis (SDRA). They introduce two additional indices of dynamic retinal vessel response to flicker and claim to be able to apply them to the assessment of vessel elasticity, among other properties. For this purpose, they introduce a calculated index, the average peak ratio (APR), which is derived from the baseline diameter fluctuation (BDF) and maximum dilation response to flicker, DA (dilation amplitude): APR = DA/BDF. Unfortunately, this supposed characterization of vascular elasticity is flawed by the nature of the assessment. Vessel elasticity cannot be directly characterized by only a single constitutive assessment of reactive vessel dilation in response to flicker light and the magnitude of spontaneous vascular vasomotions. Heitmar et al. do not explain their reason for calling APR a measure of elasticity. Moreover, the results of the APR are not disclosed anywhere in the paper.

The other index introduced by Heitmar et al.,1 ΔD, represents the degree of vessel dilation that occurs between the time point of flicker initiation and maximum vascular dilation. The problem inherent in this new parameter is its arbitrary limitation to a 1-second diameter assessment before flicker initiation as its main reference point. As can be seen in Figure 1, the diameter at 1-second in the same person varies considerably and may influence the calculation based on it, but not necessarily the maximum vessel response itself (e.g., response of volunteer 1 in Fig. 1). This maximum response is dependent on the mean baseline level of the vessel diameter, as well as on the regulatory capacity of an individual vessel, rather than on the particular phase of the vasomotion at the time point of the flicker initiation (Fig. 1, volunteer 1). Considering the impact of this individual regulatory capacity on the time course of the vessel response to flicker, we would like to point out that a vessel is not capable of dilating more than a certain threshold diameter (Fig. 1, dashed gray line), which is determined by the vessel's structural organization (e.g., collagen frame and stiffness) and is independent of the baseline diameter before the stimulation, as is shown clearly in both volunteers in Figure 1. Volunteer 1 shows different vasomotion stages at flicker initiation, and volunteer 2 shows different baseline levels at flicker initiation. The failure of the vessel diameter to return to the baseline level at the beginning of the subsequent flicker in healthy subjects has been described in other publications.2,3 This fact explains the reduced vessel dilation in both the second and the third flicker episodes compared with the first one and the rationale for the significant differences between the groups found by Heitmar et al.1 for ΔD during the first flicker episode only.

Full temporal arterial diameter course measured with a standardized RVA protocol, including three consecutive flicker stimulations in two healthy volunteers. Gray thin trace: the original time course; black race: a smoothed curve obtained by a running median with a window of 5 seconds. Note that (1) the flicker stimulus may start at every phase point of the vasomotion; (2) the “remaining dilatory capacity” in volunteer 1 does not depend on the vasomotion phase at the beginning of each flicker and is similar at each cycle; and (3) the baseline values before each stimulus increased during the assessment in volunteer 2. Such a behavior is typical of healthy arterial reactions. It has been described previously elsewhere.2,3

Figure 1.

Full temporal arterial diameter course measured with a standardized RVA protocol, including three consecutive flicker stimulations in two healthy volunteers. Gray thin trace: the original time course; black race: a smoothed curve obtained by a running median with a window of 5 seconds. Note that (1) the flicker stimulus may start at every phase point of the vasomotion; (2) the “remaining dilatory capacity” in volunteer 1 does not depend on the vasomotion phase at the beginning of each flicker and is similar at each cycle; and (3) the baseline values before each stimulus increased during the assessment in volunteer 2. Such a behavior is typical of healthy arterial reactions. It has been described previously elsewhere.2,3

The reference point of 1 second before the flicker initiation assigns an arbitrary diameter that occurs during the vasomotor diameter oscillation in each person. Since individual peaks or troughs within the oscillating diameter are smoothed out by the statistical calculations, Heitmar et al.1 found an average maximum dilation in response to flicker relative to the baseline mean for a flicker episode in a group of subjects with similar pathophysiologic status. Indeed when the group mean is calculated, the uncertainty of the 1-second reference will be compensated for, and no significant difference between the authors' method of assessing ΔD and the reference to the baseline mean in the retinal vessel analyzer (e.g., RVA v. 4.15; IMEDOS Systems UG, Jena, Germany) software is likely to be found.

The introduction of a separate analysis for each flicker episode proposed by Heitmar et al.1 seems to be an interesting option, but it has several limitations. To begin with, it triples the number of analyzed parameters, reducing thereby the level of statistical significance. Furthermore, the basic baseline level changes spontaneously, not only with vasomotions, but also undergoes changes at much lower frequencies, due to the overlap of metabolic and endothelial regulation4 or the circadian rhythm. Depending on the basic baseline level before the flicker simulation, a healthy retinal vessel will behave either according to the scenario exemplified by volunteer 2 (i.e., the relatively predilated vessel) or similar to the scenario exemplified by volunteer 1 (i.e., relatively preconstricted vessel) (Fig. 1). Both volunteers were healthy and possessed a comparable normal regulatory capacity. An averaged response from three flicker episodes would show similar vessel dilations in both subjects. However, an arbitrary choice of a separate flicker response episode to compare would denote that the vessel in volunteer 1 dilated more or less than that in volunteer 2. Which flicker episode should therefore be chosen for comparison? Smokers in the study by Heitmar et al.1 demonstrated significant differences from the control group in the first flicker episode. Such variation may not be the case for other diseases. Using this approach on another group of healthy volunteers may demonstrate different results, despite their similar pathophysiologic status.

Finally, the reference to a single temporal time point and a single flicker episode included in the parameter ΔD is too uncertain from the standpoint of the RVA measurement technique,2,5 since a qualitative assessment of each single flicker episode is not always possible in all subjects (e.g., elderly persons, subjects with bad fixation, or those who blink during the assessment), which represent a serious limitation of the method presented by Heitmar et al.1

Therefore, our main criticism of Heitmar et al. relates to each of the two suggested indices of the SDRA method. Their supposed index APR describing vascular elasticity is arbitrary. It does not convey any information regarding vascular elasticity, and its values are not reported in the paper. Their other index, ΔD, does not add any new information for an individual subject, since its definition is arbitrary. Regarding this second index, Heitmar et al.1 discuss two additional points that supersede the standard vessel dilation assessment:

Their method supports the necessity of assessing the maximum dilation at the time point of its occurrence rather than at the fixed time point of flicker cessation. This aspect has been described and discussed elsewhere.3,6

It shows a possible higher rate of dilation in the first flicker episode in the group of healthy subjects, compared with subsequent episodes, and in the first flicker episode in smokers. In a healthy population, this effect has been described in other publications.2,3 Since dilation depends on the baseline state, which is not always the same in all the flicker episodes, the value of this finding is debatable.

We would like to point out that it is worthwhile to interpret vessel diameter changes and their dynamics, which include superimposed systemic and local effects caused by cardiac pulsations, breathing rhythms, blood pressure waves and vasomotion, among others,2,4,7 and result in a different measured diameter at any given time point. Heitmar et al.1 acknowledge this fact and integrate it into their calculations by using BDF, which we think may be an important parameter to consider for the assessment of vascular health in the future.

Full temporal arterial diameter course measured with a standardized RVA protocol, including three consecutive flicker stimulations in two healthy volunteers. Gray thin trace: the original time course; black race: a smoothed curve obtained by a running median with a window of 5 seconds. Note that (1) the flicker stimulus may start at every phase point of the vasomotion; (2) the “remaining dilatory capacity” in volunteer 1 does not depend on the vasomotion phase at the beginning of each flicker and is similar at each cycle; and (3) the baseline values before each stimulus increased during the assessment in volunteer 2. Such a behavior is typical of healthy arterial reactions. It has been described previously elsewhere.2,3

Figure 1.

Full temporal arterial diameter course measured with a standardized RVA protocol, including three consecutive flicker stimulations in two healthy volunteers. Gray thin trace: the original time course; black race: a smoothed curve obtained by a running median with a window of 5 seconds. Note that (1) the flicker stimulus may start at every phase point of the vasomotion; (2) the “remaining dilatory capacity” in volunteer 1 does not depend on the vasomotion phase at the beginning of each flicker and is similar at each cycle; and (3) the baseline values before each stimulus increased during the assessment in volunteer 2. Such a behavior is typical of healthy arterial reactions. It has been described previously elsewhere.2,3